Preservation of tissues from surgical procedures is currently a topic of great importance. Currently, there are no standard procedures for fixing tissues and this lack of organization leads to a variety of staining issues both with primary and advanced stains. The first step after removal of a tissue sample from a subject is to place the sample in a liquid that will suspend the metabolic activities of the cells. This process is commonly referred to as “fixation” and can be accomplished by several different types of liquids. The most common fixative in use by anatomical pathology labs is 10% neutral buffered formalin (NBF). This fixative forms cross-links between formaldehyde molecules and amine containing cellular molecules. In addition, this type of fixative preserves proteins for storage.
When used at room temperature, NBF diffuses into a tissue section and cross-links proteins and nucleic acids, thereby halting metabolism, preserving biomolecules, and readying the tissue for paraffin wax infiltration. The formalin can be at slightly elevated temperature (i.e., higher than room temperature) to further increase the cross-linking rate, whereas lower temperature formalin can significantly decrease the cross-linking rate. For this reason, histologists typically perform tissue fixation at room temperature or higher. Some groups have used cold formalin, but only in specialized situations and not for fixing tissues. For instance, cold formalin has been used to examine lipid droplets.
Several effects are often observed in tissues that are either under exposed or over exposed to formalin. If formalin has not diffused properly through the tissue samples, outer regions of the tissue samples exposed to formalin may be over-fixed and interior regions of the tissue samples not exposed to formalin may be under-fixed, resulting in very poor tissue morphology. In under-fixed tissue, subsequent exposure to ethanol often shrinks the cellular structures and condenses nuclei since the tissues will not have the chance to form a proper cross-linked lattice. When under-fixed tissue is stained, such as with hematoxylin and eosin (H&E), many white spaces may be observed between the cells and tissue structures, nuclei may be condensed, and samples may appear pink and unbalanced with the hematoxylin stain. Tissues that have been exposed to excess amounts of formalin or too long typically do not work well for subsequent immunohistochemical processes, presumably because of nucleic acid and/or protein denaturation and degradation. As a result, the optimal antigen retrieval conditions for these tissues do not work properly and therefore the tissue samples appear to be under stained.
Proper medical diagnosis and patient safety often require properly fixing the tissue samples prior to staining. Accordingly, guidelines have been established by oncologists and pathologists for proper fixation of tissue samples. For example, according to the American Society of Clinical Oncology (ASCO), the current guideline for fixation time in neutral buffered formalin solution for HER2 immunohistochemistry analysis is at least 6 hours, preferably more, and up to 72 hours. It may be advantageous to develop a process for monitoring diffusion of fixatives through a tissue sample to determine whether the fixative has infused the entire tissue sample to minimize or limit under-fixed tissue or over-fixed tissue and to better preserve biological molecules, tissue morphology, and/or post-translational modification signals before significant degradation occurs.
Overveiw of Disclosure
At least some embodiments disclosed herein are methods of preparing specimens for a fixation process. Specimens, such as solid tissue samples, can be contacted with a liquid fixative that travels through the tissue samples. The fixative can be allowed to diffuse throughout substantially the entire thickness of the tissue samples. After a sufficient amount of fixative has infused the tissue samples, a fixation process can be performed to fix substantially all of the tissue, thereby minimizing or limiting under-fixation and over-fixation in, for example, outer regions and inner regions. The method can enhance the quality in preservation of tissue methodology, protein structure, and/or post-translation modification signals.
At least some embodiments disclosed herein are methods and systems for analyzing a tissue sample based on its characteristics, including acoustic characteristics, mechanical characteristics, optical characteristics, or other characteristics that may be static or dynamic during processing. In some embodiments, acoustic properties of the tissue sample are continuously or periodically monitored to evaluate the state and condition of the tissue sample throughout processing. Based on the obtained information, processing can be controlled to enhance processing consistency, reduce processing times, improve processing quality, or the like.
Acoustics can be used to non-invasively analyze tissue samples. When an acoustical signal interacts with tissue, transmitted signals (e.g., signals transmitted through the tissue sample) depends on several mechanical properties of the sample, such as elasticity and firmness. The acoustic properties of tissue samples may change as liquid reagent (e.g., a liquid fixative) travels through the sample. In some procedures, acoustic properties of the tissue sample can change as interstitial fluid is displaced with liquid reagent because of different acoustic properties between the interstitial fluid and liquid reagent. Even though fixatives may not result in substantial cross-linking, the acoustic properties of the tissue sample can change as the fixative travels across the thickness of the sample. The sample's acoustic properties can change during, for example, a pre-soak process (e.g., diffusion of cold fixative), a fixation process, a staining process, or the like. In the fixation process (e.g., a cross-linking process), the speed of transmission of acoustic energy can change as the tissue sample becomes more heavily cross-linked.
In some embodiments, a method for tissue preparation can include contacting a tissue sample with a fixative. Real-time monitoring can be used to accurately track movement of the fixative through the sample. After the fixative has diffused through the tissue sample, a fixation process and a subsequent histological process can be performed. A status of a biological sample can be monitored based on a time of flight of acoustic waves. The status can be a diffusion status, a density status, a fixation status, a staining status, or the like. Monitoring can include, without limitation, measuring changes in a level of diffusion, sample density, cross-linking, decalcification, stain coloration, or the like. The biological sample can be solid or non-fluidic tissue, such as bone, or other type of tissue.
In some embodiments, methods and systems are directed to using acoustic energy to monitor a tissue sample. Based on interaction between the acoustic energy in reflected and/or transmission modes, information about the specimen may be obtained. Examples of measurements include acoustic signal amplitude, attenuation, scatter, absorption, time of flight (TOF) in the specimen, phase shifts of acoustic waves, or combinations thereof. In some procedures, a fixative is applied to the specimen. As the fixative diffuses through the specimen, the tissue sample's mechanical properties (e.g., elasticity, stiffness, etc.) change, and these changes can be monitored using sound speed measurements via TOF. Subsequent processes can be monitored based on TOF and a state of the specimen (e.g., a state of saturation, a fixative state, a histological state, etc.) can be determined. To avoid under-fixation or over-fixation, the static characteristics of the tissue (including reagent(s) in the tissue), dynamic characteristics of the tissue, or both can be monitored. Characteristics of the tissue include transmission characteristics, reflectance characteristics, absorption characteristics, attenuation characteristics, or the like.
In some procedures, an unfixed tissue sample is contacted with a fixative. The movement of the fixative through the tissue sample can be monitored in real-time. The composition of the fixative can be selected to enhance monitoring. For example, NBF has a relatively high bulk modulus compared to interstitial fluid. The sound transmissibility of the fixative is related to its bulk modulus (k) and density (p) according to the speed equation,
      speed    ⁢                  ⁢    of    ⁢                  ⁢    sound    ⁢                  ⁢    in    ⁢                  ⁢    fixative    =                    k        p              .  The fixative, such as formalin, with a bulk modulus greater than interstitial fluid can significantly alter the TOF as it displaces the interstitial fluid. Once a desired level of diffusion is achieved, the tissue sample can be removed from the fixative to generally stop further diffusion. The tissue sample, including the fixative within the tissue sample, can be heated to promote cross-linking and fixation.
A TOF acquisition scheme can be used to monitor tissue samples. The TOF acquisition scheme can include an ND conversion scheme (e.g., about 1 μsec phase comparison) to obtain a large number of phase comparisons to provide generally real-time monitoring. The phase comparisons can be performed at the same frequency and phase relationship, and the temperature of the fixative and/or tissue sample can remain generally constant to increase signal to noise ratios. Because fluctuations in temperature may cause measureable changes in TOF, the TOF acquisition scheme can compensate for changes in TOF attributable to, for example, temperature changes.
In some embodiments, a method for evaluating tissue sample includes contacting tissue sample with a reagent. Diffusion of the reagent through the tissue sample can be monitored based on properties of the tissue sample, including mechanical properties, acoustic properties, and/or optical properties. Monitoring includes, without limitation, measuring time of flight of acoustic waves that travel through a tissue sample. This monitoring can include, transmitting acoustic waves across a thickness of the sample while a reagent gradually moves across the tissue sample. After the reagent in the form of fixative has diffused through most of the volume of the tissue sample, a fixation process is performed. For example, the fixative can diffuse through at least 90% by volume of the tissue sample. In other embodiments, the fixative can diffuse through substantially all the volume of the tissue sample. In one procedure, the fixative can diffuse through at least 95% by volume of the tissue sample. Such processes can substantially eliminate over-fixation or under-fixation of interior and outer regions of tissue samples.
In some embodiments, a processing method comprises contacting a tissue sample, which is in an unfixed state, with a liquid fixative. The movement of the liquid fixative through a tissue sample is acoustically monitored. A fixation process can be performed after the liquid fixative has displaced a target volume of interstitial fluid from the tissue sample. In one procedure, the fixation process is performed after the liquid fixative has displaced at least 50% by volume of the interstitial fluid. In other procedures, the fixation process is performed after the fixative has displaced at least 75% by volume of the interstitial fluid. Such fixation processes can include heating the tissue sample to promote cross-linking.
A processing system, in some embodiments, comprises an acoustic monitoring device and a computing device communicatively coupled to the acoustic monitoring device. The acoustic monitoring device can detect acoustic waves that have traveled through a tissue sample. The computing device can be configured to evaluate the speed of an acoustic wave traveling through the tissue sample based on time of flight. The computing device, in some embodiments, includes instructions for monitoring diffusion of a liquid using the acoustic monitoring device and for performing a fixation process. The acoustic monitoring device, in some embodiments, includes one or more transmitters and one or more receivers. The tissue sample can be immersed in a liquid fixative while the transmitters and receivers communicate to detect time of flight of acoustic waves.
In some embodiments, a method of evaluating a tissue sample comprises contacting the tissue sample with a fixative. The movement of the fixative through the tissue sample can be monitored based on the acoustic waves. In certain procedures, the monitoring can include simultaneously monitoring diffusion and cross-linking performed at the same or different processing temperatures. For example, diffusion and cross-linking can be performed while a fixative and/or a tissue sample is maintained at the same general temperature. In other procedures, the diffusion and cross-linking can be performed at different temperatures.